Received April 26, 2002
NAD+-dependent formate dehydrogenases (EC 1.2.1.2, FDH) of
methylotrophic bacteria Pseudomonas sp. 101 (PseFDH) and
Mycobacterium vaccae N10 (MycFDH) exhibit high homology. They
differ in two amino acid residues only among a total of 400, i.e.,
Ile35 and Glu61 in MycFDH substitute for Thr35 and Lys61 as in PseFDH.
However, the rate constant for MycFDH thermal inactivation in the
temperature range of 54-65°C is 4-6-times higher than the
corresponding rate constant for the enzyme from Pseudomonas sp.
101. To clarify the role of these residues in FDH stability the
dependence of the apparent rate constant for enzyme inactivation on
phosphate concentration was studied. Kinetic and thermodynamic
parameters for thermal inactivation were obtained for both recombinant
wild-type and mutant forms, i.e., MycFDH Glu61Gln, Glu61Pro, Glu61Lys
and PseFDH Lys61Arg. It has been shown that the lower stability of
MycFDH compared to that of PseFDH is caused mainly by electrostatic
repulsion between Asp43 and Glu61 residues. Replacement of Lys61 with
an Arg residue in the PseFDH molecule does not result in an increase in
stability.
KEY WORDS: formate dehydrogenase, thermal inactivation, thermal
stability, directed mutagenesis, Pseudomonas sp. 101,
Mycobacterium vaccae N10

NAD+-dependent formate dehydrogenases (EC 1.2.1.2, FDH),
being composed of two identical subunits and not containing metal ions
or prosthetic groups in the active site, present a unique group among
the enzymes catalyzing formate-ion oxidation to CO2 in the
cell. Formate dehydrogenases of this group are widespread in nature
(for details see [1, 2]) and
belong to the superfamily of D-specific NAD(P)+-dependent
dehydrogenases of 2-hydroxy acids [3]. FDHs of this
type have been found in all strains of methanol-utilizing yeast of
Candida, Hansenula, and Pichia species [1]. In contrast, this type of FDH is rather rare among
methylotrophic bacteria, and currently only five strains synthesizing
this type of FDH are known, i.e., Pseudomonas sp. 101 [4], Moraxella C-1 [5],
Paracoccus sp. 12A [6], Mycobacterium
vaccae N10 [7], and Hyphomicrobium sp.
JC17 [8]. The genes of all these enzymes have been
cloned and expressed in E. coli cells [7-10]. In addition, the genes of bacterial FDHs were
found in the genome of uncultured proteobacterium EBAC31A08 (EMBL
Accession AF279106) and in pSymA megaplasmid of symbiotic
nitrogen-fixing bacterium Sinorhizobium meliloti [11]. The complete gene sequences for FDHs of
methylotrophic yeast Pichia angusta (previously named
Hansenula polymorpha) [12], Candida
methylica [13], Candida boidinii [14, 15], baker's yeast
Saccharomyces cerevisiae (EMBL Accession Z75296), fungi
Aspergillus nidulans [16], Neurospora
crassa [17], potato mitochondria [18], and barley [19] have been
recently established.

All FDHs are characterized by similar values of Km for
formate and NAD+ [2]. However, the
specific activity of bacterial FDHs is about 1.5-2-times higher than
that of eucaryotic FDHs. The second feature of bacterial FDHs
distinguishing them from analogous enzymes of other sources is their
higher thermal stability. Pseudomonas sp. 101 FDH can be stored
at 4°C in phosphate buffer, pH 7.0, for a couple of years
without changing its activity. At the same time, the commercially
available lyophilized preparations of FDH from C. boidinii yeast
(Boehringer Mannheim, Germany, purity <50%), containing special
stabilizing additives, loses 50% of the initial activity in two weeks
being dissolved in phosphate buffer and stored at 4°C. The period
of half-inactivation of highly purified preparations of the above
enzyme is even lower, no more than 2-3 days [15].

Comparison of amino acid sequences of FDHs from different sources (Fig.
1) shows that the bacterial enzymes contain a
29-residue longer N-terminal region. In accordance with the data of
X-ray analysis [20], this region represents a long
non-structured loop (Fig. 2). One may assume that
the interaction of amino acid residues of this loop with the amino acid
residues of the rest of the protein globule is one of the reasons for
higher stability of bacterial FDHs. FDH from M. vaccae N10
(MycFDH), whose gene was cloned earlier, is the optimal one to test the
above hypothesis [7]. This enzyme differs from FDH
of Pseudomonas sp. 101 (PseFDH) by two substitutions only, i.e.,
Ile35 for Thr and Glu61 for Lys, but the rate of thermal inactivation
of MycFDH is 4-6-times higher than that of PseFDH. The first of the
above residues is located directly in the loop (Fig. 1), and the second one is neighboring Asp43 (Fig. 2) located in the loop, which is missing in
non-bacterial FDHs. The mutant forms of MycFDH with the Glu61Lys,
Glu61Gln, and Glu61Pro replacements were obtained earlier [7]. The preliminary studies on the thermal
inactivation of these mutants at 60.5°C demonstrated their higher
stability [7]. However, the comparison of rate
constants for thermal inactivation at one fixed temperature cannot be
used as a characteristic of the stabilization effect because the
temperature dependence of these rate constants for the wild-type and
mutant forms may be completely different. To exemplify the above
statement we can point to the data on the thermal stability of the
PseFDH Arg284Gln mutant: to achieve a 10-fold increase in the
inactivation rate constant for the wild-type enzyme one needs to go
4°C up (see Results), while for the noted mutant to
get the same enhancement one has to increase the temperature 25°C
up [21]. This results in a similar stability of
the wild-type and Arg284Gln PseFDH mutant at 54°C, and in a
20-fold increase in stability of the mutant compared to the wild-type
enzyme at 68°C. In addition, the temperature dependence of the
inactivation rate constant gives information on the component, i.e.,
entropy or enthalpy, predominantly determining the enzyme stability.

Fig. 2. A general view of the holo-form of Pseudomonas sp.
101 formate dehydrogenase (PDB2NAD.ENT [17]) and
the enlarged fragment with Asp43 and Lys61 residues. The
non-structured loop in the sequence 11-46 is shown in
white. Oxygen atoms of carboxy-group of Asp43 and nitrogen atom of
Lys61 are shown in white balls. The picture was made using RasMol 2.6b
program.

The goal of the present work was to quantitatively estimate the effect
of amino residues in positions 35 and 61 on thermal stability of
bacterial FDH. To clarify the role of these residues in FDH stability,
the dependence of the apparent rate constant for enzyme inactivation on
phosphate concentration was studied. Kinetic and thermodynamic
parameters for thermal inactivation were obtained for both recombinant
wild-type and mutant forms, i.e., MycFDH Glu61Gln, Glu61Pro, Glu61Lys
and PseFDH Lys61Arg.

MATERIALS AND METHODS

The following enzymes were used: phage T4 DNA-polymerase
(10 U/µl), DNA-ligase (400 U/µl), and
polynucleotide kinase (16 U/µl), all from New England
Biolabs (USA). All reagents used for genetic engineering manipulations
were of molecular biology grade (Sigma, USA).

Site-directed mutagenesis was performed in accordance with the
Kunkel method as described in [7]. To confirm the
unique mutation sites the gene-containing plasmid regions were
sequenced in both directions using an Applied Biosystems model 370A
automated DNA sequencer (USA) and an ABI PRISM DNA Sequencing Kit based
on fluorescent labeled terminators produced by the same company. The
mutagenesis efficiency was about 60-100%. For the thermal stability
studies each mutant enzyme was produced using the plasmids isolated
from two individual clones.

Production and purification of MycFDH, PseFDH, and
their mutant forms. Biomass of E. coli TG1 cells with the
corresponding plasmid was produced by cultivation of a single colony in
200 ml of 2YT medium (16 g/liter bactotryptone,
10 g/liter yeast extract (both from Difco, USA), and
5 g/liter NaCl, pH 7.0) containing 150 µg/ml ampicillin for
12-15 h at 37°C. The inducer of FDH biosynthesis,
beta-isopropyl-D-thiogalactoside (IPTG), was added in the
beginning of cultivation up to 0.5 mM. The cells were collected by
centrifugation at 8000g on a Beckman J-21 centrifuge (USA) for
10 min. The further purification of MycFDH and PseFDH and their
mutant forms was performed using the standard protocol developed for
the recombinant FDH of Pseudomonas sp. 101 expressed in E.
coli [22]. The protocol included cell
disruption in a ultrasonic disintegrator, ammonium sulfate
fractionation (40% saturation) and FPLC hydrophobic chromatography
(Pharmacia Biotech, Sweden) on a 1 × 10 cm column packed
with highly substituted Phenyl Sepharose Fast Flow (Pharmacia Biotech).
The enzyme preparations obtained were at least 90-95% purity as judged
from SDS-PAGE. For the purposes of thermal stability studies, all
enzyme preparations were transferred into a K-phosphate buffer of the
needed concentration, pH 7.0, by gel filtration through Sephadex
G-25 (Pharmacia Biotech).

For each individual enzyme four independent preparations from four
biomass samples were obtained.

The dependence of inactivation rate on phosphate buffer concentration
was studied at 61°C in the concentration range of 0.01-1.26 M.
The temperature dependence of the inactivation rate constant was
studied in the temperature range 54-65°C in 0.1 M phosphate
buffer. Enzyme solution (50 µl) dissolved in 0.1 M
K-phosphate buffer, pH 7.0, preliminary heated to 45°C, was added
to 550 µl of the same buffer, preliminary heated to the needed
temperature (with the accuracy of ±0.1°C), to the final
enzyme concentration of 0.05-0.1 mg/ml, vigorously shaken, and
incubated at the designated temperature. To measure the enzyme residual
activity 50 µl aliquots were taken at fixed intervals. The
thermal inactivation rate constant kin was determined
from the time course of enzyme inactivation treated as first-order
kinetics by nonlinear regression using the Origin 4.1 program. Each
value of kin for the native and mutant FDH forms
represents an average from at least four independent measurements. The
activation parameter DeltaH. of thermal inactivation
process was determined as a slope of a ln(kin/T)
versus 1/T plot using linear regression with the statistic weights of
(kin/T)2/s2, where T is
the absolute temperature, and s is the error of
kin determination calculated from primary plots. The
value of DeltaS. was determined from the slope of the
DeltaG. versus T plot.

Modeling and structural optimization of FDH mutants was performed with
the Insight II program on a Silicon Graphics workstation. To visualize
and analyze protein three-dimensional structures RasMol 2.6b and WebLab
Viewer Pro 3.7 (MSI) programs were used.

RESULTS

To clarify the role of amino acid residues in positions 35 and 61 in the
sequence of bacterial FDH in the enzyme thermal stability the
physicochemical characteristics of the inactivation process were
studied for the wild-type MycFDH and PseFDH and their single point
mutants: MycFDH Glu61Gln, Glu61Pro, Glu61Lys and PseFDH Lys61Arg. The
first mutation, Glu61Gln, removes the negative charge, and Glu61Pro
mutation provides both the removal of the negative charge and the
increase in the rigidity of the polypeptide chain. Glu61Lys mutant of
MycFDH and wild-type PseFDH differ in only one amino acid residue in
position 35, the former has Ile, and the latter has Thr. The comparison
of inactivation rates of these enzymes allows one to determine the
contribution of the Thr35Ile replacement to the enzyme stability. The
construction of PseFDH Lys61Arg mutant was aimed to further improve the
thermal stability of the enzyme. The improvement of stability could
originate from the facts that: 1) a guanidinium group of arginine
residue is a much stronger base than epsilon-amino group of the
lysine residue, and thus, the ionic pair Asp43-Arg61 will be stronger
than Asp43-Lys61; and 2) comparative statistical analysis of structures
of proteins from mesophylic and thermophylic microorganisms [24] demonstrates the presence of Arg residue instead
of Lys in alpha-helix regions of enzymes with high thermal
stability.

The time course of wild-type and mutant FDHs inactivation at 60°C
is presented in Fig. 3. As one can see from these
data, the time dependence of enzyme residual activity is linear in
semi-logarithmic plots (lnA versus t). The value of the slope of
the straight lines, i.e., the apparent inactivation rate constant
kin, was independent of enzyme concentration in the
range of 0.03-0.25 mg/ml. The linear character of dependences in
Fig. 3 and the independence of the apparent rate
constants on the initial enzyme concentration indicate that FDH thermal
inactivation proceeds in accordance with first order kinetics. In other
words, this indicated the unfolding of the protein globule is not
preceded by enzyme dissociation into individual active subunits. It
must be noted that PseFDH is known for strong interaction between its
subunits. For instance, the application of 8 M urea is not
sufficient to dissociate the enzyme into individual subunits for the
purpose of specific modification of Cys145 residue located in the
region of the inter-subunit contact. The data presented in Fig. 3 prove the important role of the residue in position
61 in the enzyme stability. The lowest stability is observed for the
wild-type MycFDH. The removal of the negative charge (Glu61Gln
mutation) improves the thermal stability decreasing the inactivation
rate constant by 2.5-fold. Moreover, introduction of a positive charge
of Lys residue in this position, Glu61Lys mutant of MycFDH, yields an
enzyme whose stability is close to that of the wild-type PseFDH. A
comparable stabilization effect is achieved by introducing a proline
residue in position 61 (Fig. 3). The replacement
Lys61Arg in the PseFDH molecule did not lead to stability
improvement.

Fig. 3. Time course of thermal inactivation of wild-type and
mutant FDHs from M. vaccae N10 and Pseudomonas sp. 101 at
60°C. Since the pairs of enzymes MycFDH Glu61Lys-MycFDH Glu61Pro
and wild-type PseFDH-PseFDH Lys61Arg show very close stability, their
inactivation curve is shown as one straight line. Enzyme concentration
0.05-0.1 mg/ml, 0.1 M K-phosphate buffer,
pH 7.0.

The efficiency of electrostatic interaction significantly drops with an
increase in the ionic strength. To discriminate between the
contribution of residues in positions 35 and 61 into the FDH stability
the dependence of the apparent rate constant for the enzyme
inactivation on the concentration of phosphate buffer was studied. The
choice of phosphate for the purpose of ionic strength studies was based
on the big radius of its anion. The latter prevents phosphate from
penetrating inside the protein globule, and thus, phosphate can affect
only ion pairs located at the surface of the enzyme molecule. The
dependence of the apparent rate constants for the studied enzymes on
phosphate concentration in the range 0.01-1.26 M is presented in Fig.
4. The obtained bell-shaped curve is typical for
most proteins. The initial increase in ionic strength causes enzyme
destabilization and an increase in inactivation rate constant because
of the decreased efficiency of electrostatic interactions. At some
point, all ion pairs on the protein surface are destroyed and
electrostatic interaction makes no more contribution to the enzyme
stability. This leads to the maximum destabilizing effect. The further
increase in the ionic strength enhances hydrophobic interactions, and
this stabilizes FDH. We note here the three most important and
interesting results.

1. Maximum destabilization effect for the wild-type MycFDH is observed
at phosphate concentration of 0.1 M, while for all mutant forms of this
enzyme and for PseFDH the maximum decrease in stability occurs at
0.2-0.3 M concentrations.

2. Maximum 3-fold decrease of enzyme stability (expressed as the ratio
of inactivation rate constants at 0.01 and 0.21 M phosphate) is
observed for PseFDH and MycFDH E61K, which contain the ion pair
Asp43-Lys61. In the case when the above mutation is absent, for
instance in MycFDH Glu61Pro and Glu61Gln mutants, the destabilization
effect is smaller and is equal to 2. The minimum decrease in stability
(1.4-fold) is observed for the wild-type MycFDH because an increase in
the ionic strength diminishes the repulsion of carboxy-groups of Asp43
and Glu61.

3. At high phosphate concentrations destroying all surface ion pairs,
the rate constants for the wild-type MycFDH and all its mutant forms
are equal and only 1.5-fold higher than kin for
PseFDH. This means that, under the above conditions, the difference in
stability of PseFDH and all MycFDH forms is based only on the nature of
the amino acid residue in position 35.

To get the complete information on the mechanism of wild-type and mutant
FDH inactivation the temperature dependence of the apparent rate
constants for enzyme inactivation was studied in the 54-65°C range
(Table 1). To conduct these experiments we have
chosen the concentration of phosphate buffer equal to 0.1 M since
it is the concentration which provides the maximum difference in
stability of wild-type and mutant MycFDHs and PseFDH. The data
presented in Table 1 indicate the different
effect of temperature on the inactivation rates of the studied enzymes.
We note that the increase in rigidity of the polypeptide chain caused
by the replacement of Glu61 with Pro provides the enzyme with a higher
stability at 62°C than the restoration of the ionic pair
Asp43-Lys61 (Table 1). Different temperature
dependence is also observed for the wild-type PseFDH (ion pair
Asp43-Lys61) and its K61R mutant (ion pair Asp43-Arg61). At 54 and
60°C, a slight but reliable and repeatedly reproducible increase
in stability of PseFDH K61R mutant compared to the wild-type PseFDH is
observed, while at 62°C and above the wild-type enzyme is more
stable (Table 1).

The secondary plots of ln(kin/T) dependence on 1/T
were linear. The linear character of the secondary plots proves that
thermal inactivation of wild-type and mutant FDH forms can be described
by the theory of an activated complex. In accordance with the latter
[25], the temperature dependence of the apparent
rate constant for thermal inactivation, kin, is
presented by the following equation:

where k and h are the Boltzmann and Plank constants,
respectively, R is the universal gas constant, and
DeltaH. and DeltaS. are the
activation parameters of thermal inactivation. The values of
DeltaH. and DeltaS. can be
calculated from slopes and intercepts of the secondary plots
ln(kin/T) versus 1/T, respectively. However, the
extrapolation of the linear dependence to the ordinate axis to
determine the value of DeltaS. gives a high error in
its determination. The more precise value for this parameter can be
obtained from the dependence of DeltaG. on T (Fig. 5). The calculated values of DeltaH.
and DeltaS. are shown in Table 2. We note that the conversion from PseFDH to MycFDH
is accompanied by changes in both DeltaH. and
DeltaS., and moreover, the change in
DeltaH. value is ~60 kJ/mol or 15%.

The data obtained in this study confirm the important role of the
non-structured loop in the region of 15-43 residues in providing high
thermal stability of bacterial FDHs. The replacement of Lys61 residue
with Glu residue in the MycFDH molecule results in a significant drop
in the enzyme stability. The data on thermal stability of Glu61Gln and
Glu61Lys mutants of MycFDH indicate the importance of electrostatic
interaction between the residues in positions 43 and 61. The removal of
the negative charge in position 61 in the first mutant results in
slight stabilization of the enzyme, while the restoration of the ionic
pair Asp43-Lys61 yields the MycFDH mutant, whose rate constant of
thermal inactivation is only 1.5-fold higher that the corresponding
rate constant for PseFDH (Table 1). The
destabilization of MycFDH compared to PseFDH is partially due to the
mutation of residue 35 (Ile35Thr). The side group of residue 35 is
exposed to the solution, and the replacement of the hydrophilic side
group of Thr residue with a bulky hydrophobic side group of Ile can
worsen the entropy. As shown in our earlier works on Ser-Ala mutations
in alpha-helix regions of PseFDH [26], the
entropy change originating from the replacement of a polar group with a
nonpolar one results in a 9-40% change in the enzyme stability.
Nevertheless, the contribution of this mutation is much smaller
compared to introduction of glutamic acid to replace lysine at position
61.

The observed changes in thermal stability in our experiments are rather
small compared to the effects that could be predicted for optimized
electrostatic interactions [20]. The data on X-ray
structural analysis for both apo- and holo-forms of PseFDH confirm the
total exposure of the Asp43 carboxy-group and the Lys61 amino group
into solution (Fig. 2). Thus, the efficient
interaction between two oppositely charged groups is strongly weakened
because of the high dielectric constant of water. This may explain the
absence of additional stabilization in the case of Lys61Arg replacement
in PseFDH.

As noted earlier, bacterial FDHs possess an additional 29-residue
sequence in the N-terminal region (Fig. 1), which
forms a non-structured loop (Fig. 2, shown in gray
color) absent in the eukaryotic enzymes. However, the term
non-structured was used only to emphasize the formal
absence of typical elements of secondary structure. In reality, the
loop must be of high rigidity, because it has seven Pro residues among
total 35 (11-46 in sequence). Thus, this region of the amino acid
sequence of bacterial FDH is divided into small blocks of 4-7 amino
acid residues by proline residues. The analysis of thermal stability
changes induced by Glu61Pro mutation (Table 1)
leads to the conclusion that electrostatic interactions between 43 and
61 residues may be necessary for the fixation of the polypeptide chain
in the region of residue 61 but not for the stabilization of the
already rigid non-structured loop formed by residues 11-46.
The Lys61 residue is the second one in the alpha1-helix (Figs.
1 and 2), and based on general
considerations, its mutation to Pro (Lys61Pro) had to change the helix
conformation. However, the results of computer modeling for this mutant
structure demonstrate no distortion of the alpha1-helix with the
introduced mutation. At first sight this may seem unexpected; however,
a similar situation is observed for PseFDH in the case of Pro105
residue located at the second position in the alpha3-helix [20] (not shown in Fig. 1). A
similar stability of Glu61Pro and Glu61Gln mutants of MycFDH (Fig. 3, Table 1) is a strong argument
supporting the above assumption.

In conclusion, the experiments presented in this work have shown that
the non-structured loop in the region of residues 11-46,
which is present in all bacterial formate dehydrogenases and absent in
analogous eukaryotic enzymes, is one of the reasons for the higher
stability of bacterial enzymes. The data obtained demonstrate that one
of the weak points in the structure of bacterial FDHs is
the region of the polypeptide chain including residue 61. This region
can be stabilized both by electrostatic interaction with Asp43, located
in the non-structured loop and by increasing the rigidity
of the polypeptide chain and introducing Pro residue in position 61. We
are currently working on the increase in thermal stability of
Pseudomonas sp. 101 FDH, which is the most stable enzyme among
the NAD+-dependent formate dehydrogenase family. A number of
site-directed mutations have yield the enzymes with the thermal
stability prevailing over that of the wild-type enzyme by a factor of
10-20. These experiments will be reported in detail in our further
publications.

This work was supported by grants of the Russian Foundation for Basic
Research 99-04-49156 and 02-04-49415.